Polynucleotide phosphorylase was used to covalently link poly(A) tracts, having 5,18,31,41,59,73,100 and 150 residues to the 3' termini of E. coli (3H)rRNA(5S(3H)rRNa.(A)n). Upon hydrolysis of these polyadenylated polynucleotides by ribonucleases from Citrobacter sp., Enterobacter sp., bovine pancreas, human spleen and human plasma, it was found that the (A)n segment inhibited RNase activity, and that the inhibition was enhanced as the polyadenylic acid tract was lengthened. Certain polyamines, and polyamine analogs, restore enzyme activity in the presence of 5S rRNa.(A)n. Changing the ionic strength caused fluctuations in the amount of enzyme inhibition, as did alterations in either reaction temperature of quantities of substrate and enzyme. In all systems studied, inhibition of RNase activity mediated by 5S rRNA.(A)n may be closely regulated by the chain length of the poly(A) segment and the concentrations of the components of the reaction mixture noted above. Furthermore, use of poly(A) covalently linked to substrate, as 5S rRNA.(A)n, frequently resulted in greater inhibitions than if equal quantities of free poly(A) were used. With several enzymes, in the absence of inhibition, the substrate portion of 5S rRNA.(A)n was hydrolyzed more rapidly than the same amount of 5S rRNa, implying that the structure of 5S rRNA is altered upon covalent attachment of poly(A).